JP2012510291A - Novel Lactobacillus plantarum and composition containing the same - Google Patents

Abstract

The present invention provides a Lactobacillus plant arum CJLP56 (CJLP56) KCTC 11402BP, a composition for treating intestinal diseases containing the lactic acid bacterium, and an immune enhancing composition containing the lactic acid bacterium.
[Selection] Figure 1

Description

  The present invention relates to a novel Lactobacillus plantarum and a composition containing the same, and more particularly to a novel Lactobacillus plantarum useful for the prevention and treatment of intestinal diseases and immune diseases and a composition containing the same. Thing.

  Lactic acid bacteria, which are abundant in traditional fermented foods such as kimchi, are in charge of the role of breaking down fiber and complex proteins and making them with important nutritional components while coexisting with the digestive world of the human body. Probiotics are commonly referred to as living microorganisms that have beneficial effects on the health of the host by improving the intestinal microbial environment of the host in the gastrointestinal tract of the contained animal. In order to be effective as probiotics, it must be taken orally, reach the small intestine and remain attached to the intestinal surface, so it is basically acid resistant, bile acid resistant, and intestinal skin Cell attachment ability must be excellent.

Lactobacillus sp. Lactic acid bacteria are typical probiotics found in traditional fermented foods such as kimchi. Lactobacillus microorganisms can often be seen in the intestinal tract of animals including humans as the lactobacilli that have the same or different forms of action, and in the process of action of dairy products and vegetables. Lactobacillus microorganisms not only improve the diarrhea and constipation by maintaining the intestinal pH acidic and suppressing the growth of the same harmful bacteria as E. coli and Clostridium. It is known to play a role such as cancer action and serum cholesterol lowering. Acidophillin produced by Lactobacillus is known to inhibit the growth of Shigella, Salmonella, Staphylococcus and Escherichia coli. Moreover, it acts to stop diarrhea by suppressing the growth of diarrhea-causing bacteria and normalizing the intestinal bacteria total (Non-Patent Document 1 and Non-Patent Document 2).
Research that intends to develop live fungi and livestock feed using the aforementioned characteristics of Lactobacillus microorganisms is actively underway. Bacterial diarrhea in livestock induces decreased body weight gain and moribundity. Therefore, it has been widely practiced to add antibiotics to feed to prevent this and increase livestock production. However, due to the emergence of antibiotic-resistant bacteria and problems such as residual antibiotics in livestock products, organic livestock feeding methods tend to be emphasized by regulating the use of antibiotics in feed (Patent Document 1). (Non-Patent Document 3).

  The same lactic acid bacteria as Lactobacillus microorganisms are also known to have an immune enhancing effect. Allergies and atopy diseases related to more than the immunomodulation predicted by the influence of environmental pollution and increase of instant food intake are increasing rapidly worldwide, and such diseases are also increasing in Korea as well. It is in. Recently, in Europe, efforts have been made to alleviate or improve disease symptoms with lactic acid bacteria as part of microbial treatment (bacteriotheraphy), which treats illnesses by mouth. When Lactobacillus rhamnosus GG (Lactobacillus rhamnosus GG) was administered to infants, the incidence of atopy was reduced to a half level (Non-patent Document 4), and Lactobacillus rhamnosus and Lactobacillus rhamnosus and Lactobacillus rhamnosus When administering Bacillus reuteri (L. reuteri), there is a report that the site and degree of eczema are reduced (Non-patent Document 5).

  Research on the mechanism for the immune enhancement effect of lactic acid bacteria has been ongoing, and the specific mechanism has yet to be clearly clarified. Thus, it is known that inhabiting the intestine affects the intestinal tract immune system. For example, lactic acid bacteria intake through yogurt is known to increase antibacterial activity of lymphocytes of Peyer's patch. According to some studies conducted on laboratory animals and humans, lactic acid bacteria are , Known to enhance the reaction of IgA. Lactic acid bacteria also affect both innate and adaptive immunity. Innate immunity of the intestinal tract immune system is known to play a role in maintaining health against infection by phagocytosing and killing pathogenic bacteria. Adaptive immunity activates macrophages, which are responsible for degrading antigens and presenting them to T lymphocytes, increasing the production of various cytokines, especially interleukins IL-12 and IL-18. This is known to increase the production of cytokines by activating NF-κB and STAT signals in macrophages, which are part of lactic acid bacteria cell wall constituents. Lactic acid bacteria also increase IL-12, IL-18, and TNF-α production in the lymph nodes and dendritic cells that are present in the mucosa of the digestive system as specialized antigen-presenting cells. It is known that, together with MHC class II and B7-2, expression of surface molecules that activate T lymphocytes is also increased (Non-patent Document 6).

  T lymphocytes are the cells that play a central role in adaptive immunity, and adaptive immunity can be divided into Th1 responses of cellular immunity and Th2 responses of antibody immunity. Cytokines produced by antigen-presenting cells in Th1 and Th2 reactions differ from each other.In the Th1 reaction, IL-12, IL-18, and interferon (IFN) production predominate, and in the Th2 reaction, PGE2 IL-4 and IL-10 production is dominant. It is known that an appropriate balance should be established between the Th1 response and the Th2 response, and various immune diseases appear when the balance is broken. Th1 cells primarily fight infection, whereas Th2 cells are primarily involved in allergies and inflammatory reactions. When these function normally, Th2 cells protect the body from dust and other unwanted substances, but in the unlikely event that these cells exhibit excessive activity, IgE antibody production increases and does not threaten the human body. Causes allergic reactions to proteins (eg, pollen, food, etc.). Therefore, the Th1 and Th2 responses should be kept in balance, and if one of them is excessive or the other is insufficient, the disease is induced. Further, if cortisol is continuously released by the subsequent stress, the Th1 response decreases and the Th2 response increases, and cancer, atopy, allergies and autoimmune diseases are induced (Non-patent Document 7).

  According to in vivo experiments, lactic acid bacteria increase IFN-γ production of Th1 cytokines in T lymphocytes and suppress IL-4 and IL-5 production of Th2 cytokins (non-patent literature). 8). In other experiments, IFN-γ was increased in splenocytes when orally administered lactic acid bacteria to mice that were administered ovalbumin (Ovalbumin), a Th2-reactive animal model, and orally administered ovalbumin-primed mice. In addition, spleen splenocytes isolated in mice that had decreased IL-4, IL-5, and IgE, and were ovalbumin-administered and biased by the Th2 reaction, were orally administered with cytokines and IgE when cultured with lactic acid bacteria. It is known to be identical to the experiment. However, only T lymphocytes did not increase so that IFN-γ production was significant when cultured with lactic acid bacteria, so antigen-presenting cells such as macrophages and dendritic cells must be used for IFN-γ production of T lymphocytes. It is regarded as necessary (Non-Patent Document 9). IL-12 and IL-18, on the other hand, are produced in macrophages or award-winning cells as important cytokines for differentiating Th0 lymphocytes with Th1 lymphocytes, and are concentration-dependent if treated with spleen sputum cells or lactic acid bacteria during macrophage culture. It is known that the production of IL-12, IL-18 and IFN-α is increased. Thus, lactic acid bacteria increase the production of IL-12, IL-18, IFN-α, etc. in macrophages, thus promoting differentiation in Th1 cells and inducing these IFN-γ production, so Th2 is dominant. It acts to match the Th1 / Th2 balance in the situation (Non-Patent Document 6). Thus, lactic acid bacteria are known to be useful in preventing or treating cancer, atopy, allergies, and autoimmune diseases induced by Th1 / Th2 imbalance due to excessive Th2 response.

Korean Patent Publication 1998-78358

Michael and Philippe, Probiotics and prebiotics: Effects on diarrhea, The journal of nutrition, Volume 137, March 2007, pages 803S-811S Roberfroid, Prebiotics and probiotics: Are they functional foods, American journal of clinical nutrition, Volume 71, June 2000, pages 1682S-1687S McEwen and Fedorka-Cray, Antimicrobial use and resistance in animals, Clinical infectious Diseases, Volume 34, June 2002, pages S93-S106 Kalliomaki et.al., Probiotics in primary prevention of atopic disease: a randomized placebo-controlled trial, Lancet, Volume 357, April 2001, pages 1076-1079 Rosenfeldt et.al., Effect of probiotic Lactobacillusstrains in children with atopic dermatitis, Dermatologic and ocular diseases, Volume 111, February 2003, pages 389-395 Cross et.al., Anti-allergy properties of fermented foods: an important immunoregulatory mechanism of lactic acid bacteria, International Immunopharmacology, Volume 1, May 2001, pages 891-901 Elenkov and Chrousos, Stress hormones, Th1 / Th2 patterns, pro / anti-inflammatory cytokines and susceptibility to disease, Trends in Endocrinology and Metabolism, Volume 10, November 1999, pages 359-368 Matsuzaki et.al., The effect of oral feeding of Lactobacillus casei strain Shirota on immunoglobulin E production in mice, Journal of Dairy Science, Volume 81, January 1998, pages 48-53 Kato et.al., Lactic acid bacterium potently induces the production of interleukin-12 and interferon-gamma by mouse splenocytes, International Journal of Immunopharmacology, Volume 21, February 1999, pages 121-131 Miraglia et.al, Immune dysregulation in atopic dermatitis, Allergy and Asthma Proceedings, Volume 27, November-December 2006, pages 451-455 Remington's Pharmaceutical Sciences (19th ed., 1995) Kim et.al., Leuconostoc inhae sp. Nov., A lactic acid bacterium isolated from kimchi, International Journal of Systematic and Evolutional Microbiology, Volume 53, July 2003, pages 1123-1126 Kim et.al., Leuconostoc kimchii sp.nov., A new species from kimchi.International Journal of Systematic and Evolutional Microbiology, Volume 50, September 2000, pages 1915-1919 Kobayashi et.al., Studies on biological characteristics of Lactobacillus: II.Tolerance of the multiple antibiotic resistance strain, L.casei PSR3002, to artificial digestive fluids.Japan Journal of Microbiology.Volume 29, July 1974, pages 691-697 Casey et.al., Isolation and characterization of anti-Salmonella lactic acid bacteria from the porcine gastrointestinal tract, Letters in Applied Microbiology.Volume 39, 2004, pages 431-438 Kim et.al., Probiotic properties of Lactobacillusand Bifidobacterium strains isolated from porcine gastrointestinal tract, Applied Microbiology and Biotechnology, Volume 74, April 2007, pages 1103-1111 Hirano et.al., The effect of Lactobacillus rhamnosuson enterohemorrhagic Escherichia coliinfection of human intestinal cells in vitro, Microbiology and Immunology, Volume 47, 2003, pages 405-109 Fujiwara et.al.A double-blind trial of Lactobacillus paracasei strain KW3110 administration for immunomodulation in patients with pollen allergy, Allergology International, 2005, volume 54, pages 143-149 Fujiwara et.al., The anti-allergic effects of lactic acid bacteria are strain dependent and mediated by effects on both Th1 / Th2 cytokine expression and balance, International Archives of Allergy and Immunology, 2004, Volume135, pages 205-215

  In addition, the present inventors have conducted research to develop a new lactic acid bacterium that is very effective in regulating Th1 / Th2 imbalance caused by excessive Th2 reaction compared to the lactic acid bacterium that has been publicly announced. The present invention was completed by isolating and identifying a novel Lactobacillus acidobacillus strain.

  Therefore, the object of the present invention is not only excellent in acid resistance, bile acidity and intestinal epithelial cell adhesion ability, which are basic properties as probiotics, but also enhances immune enhancement effects, particularly Th1 / Th2 imbalance due to excessive Th2 reaction. It is to provide a novel Lactobacillus genus Lactobacillus having an excellent effect of regulating.

  Another object of the present invention is to provide a composition for preventing or treating intestinal diseases comprising the Lactobacillus strain of Lactobacillus.

  Another object of the present invention is to provide a composition for enhancing immunity comprising the Lactobacillus strain of Lactobacillus.

  In order to achieve the above object, the present invention provides Lactobacillus plantarum CJLP 56 (Deposit organization: Biobank of Biotechnology, Deposit date: 2008.10.16, Deposit number: KCTC 11402BP). .

  The present invention also provides a composition for preventing or treating intestinal diseases comprising the Lactobacillus plantarum CJLP56.

  The present invention also provides an immunopotentiating composition comprising Lactobacillus plantarum CJLP56.

  Hereinafter, the present invention will be described in more detail.

  Lactobacillus plantarum CJLP56 (Lactobacillus plantarum CJLP56) according to the present invention is a novel strain of Lactobacillus plantarum isolated and identified from traditional fermented foods. Examples of the traditional fermented food include kimchi, vegetable effect products, miso, soy sauce, neat soy sauce, and salted salt, but are not limited thereto.

  Lactobacillus plantarum CJLP56 of the present invention is the highest homology (99.9%) with the 16S rRNA nucleotide sequence analysis results Lactobacillus plantarum NBRC15891T, GenBank accession number AB326351) for sympathy and classification of microorganisms ) Showed the highest molecular phylogenetic flexibility with Lactobacillus plantarum. Therefore, the microorganism was identified by Lactobacillus plantarum, named by Lactobacillus plantarum CJLP56, and deposited at the Biotechnology Research Institute Genetic Bank on October 16, 2008 (Accession No.KCTC). 11402BP). The base sequence of the 16S rRNA gene of Lactobacillus plantarum CJLP56 seems to be the base sequence listing SEQ ID NO. 1 attached hereto.

  Lactobacillus plantarum CJLP56 of the present invention is a cultivar-positive bacterium that is capable of growing under aerobic and anaerobic conditions, is facultive anaerobe, has no motility without forming spores The cell morphology is Neisseria gonorrhoeae.

  The more specific form and physiological characteristics of Lactobacillus plantarum CJLP56 were found to be the same as Table 1 as a result of analysis by ordinary methods in the art.

  Lactobacillus plantarum CJLP56 of the present invention can be stored at -70 ° C by dissolving the cells in a storage solution prepared by mixing a certain amount of glycerol component with water in order to stably store for a long period of time. It is preferable to suspend and freeze-dry in 10% skimmed milk.

  Further, Lactobacillus plantarum CJLP56 of the present invention has a general intestinal effect and immunity enhancing effect of lactic acid bacteria as probiotics.

  In the context of the present invention, 'probiotics' is understood to mean living microorganisms that improve the intestinal microbial environment of the host in the gastrointestinal tract of animals, including humans, and have a beneficial effect on the health of the host. Is done. Probiotics are living microorganisms with probiotic activity, and are fed to the host intestinal flora when fed to humans and animals in the form of dried cells or effective products in the form of single or complex strains. Can have a beneficial effect. It is the first to be such a probiotic microorganism, confirming whether it can survive in the intestine through the stomach while being less affected by gastric juice and bile, and can settle and survive in the intestine This should have a beneficial effect on the host gut. Therefore, it must have acid resistance to gastric acid, bile acid resistance to bile acids and adhesion to intestinal epithelial cells. Second, in order to be a probiotic microorganism, there must be no problem with the safety of the microorganism, and in this connection generally gelatin liquefaction test, phenylalanine deamination production test, ammonia production Tests, hemolytic experimental tests, etc. are performed. Lactobacillus plantarum CJLP56 according to the present invention not only has excellent acid resistance, bile acid resistance to bile acid intestinal epithelial cells, but also gelatin liquefaction test, phenylalanine deamine production test and ammonia production test It was found to be safe because it was confirmed by α-hemolysis, which was determined to be unrelated to pathogenicity in experimental hemolysis.

  Lactobacillus plantarum CJLP56 according to the present invention is extremely excellent in acid resistance, bile acid resistance and enteric cell adhesion, and is expected to have an intestinal regulating effect. Therefore, in another aspect, the present invention provides a composition for preventing or treating intestinal diseases comprising Lactobacillus plantarum CJLP56.

  The composition for preventing or treating enteric diseases containing microorganisms according to the present invention is used for preventing or treating enteric diseases of mammals including humans, and preferably for domestic animals such as cattle, horses and pigs. Including. The above “intestinal diseases” include all bacterial infections and inflammatory bowel diseases because of intestines, such as infectious diarrhea, gastroenteritis, inflammatory bowel diseases, neurological enteritis caused by pathogenic microorganisms (e.g., E. coli, Salmonella, Kurosuturidium) Including, but not limited to, syndrome, small intestinal microbial overgrowth, acute intestinal diarrhea, and the like. Lactobacillus plantarum CJLP56 contained in the composition for treating intestinal diseases can be present as living cells or dead cells, but is preferably present as living cells. In general, viable cells are effective in treating and ameliorating various symptoms caused by abnormal effects of total intestinal bacteria. While preventing the fungus from colonizing, it produces lactic acid and lowers the pH in the intestine so that harmful bacteria cannot grow. In addition, the administered viable cells produce bacteriocin and peroxide to suppress the growth of hospital bacteria and help the activity of intestinal villi responsible for absorption of nutrients. In addition to this, substances that aid in the absorption and utilization of nutrients are generated to improve the feed demand rate in animals and to generate substances that neutralize toxic substances produced by pathogenic bacteria.

  The method for administering the composition for preventing or treating intestinal diseases of the present invention is not particularly limited, but it is preferably administered through the mouth. The dose varies depending on the type of intestinal disease, the degree of disease, age, sex, race, therapeutic or preventive purpose, etc. can do.

  In addition, the Lactobacillus plantarum CJLP56 of the present invention has a remarkably superior immune enhancing effect in addition to the intestinal regulating effect and other conventional lactic acid bacteria. Lactobacillus plantarum CJLP56 increases the production of IL-12 that induces a Th1 response in splenocytes and suppresses the production of IL-4 that induces a Th2 response. In addition, it stimulates immunomodulation-related cells such as macrophages and dendritic cells that regulate T cell immune responses as antigen presenting cells, and promotes the production of cytokin that induces differentiation of Th0 lymphocytes into Th1 lymphocytes. Thus, it was confirmed that there is an immunoregulatory force capable of regulating the Th1 / Th2 imbalance caused by excessive Th2 response. A more specific explanation of the immune enhancement effect of Lactobacillus plantarum CJLP56 is as follows.

  Compared to the negative control group, Lactobacillus plantarum CJLP56 was treated with Th1 response-inducing cytokin IL-12 when treated with ovalbumin (OVA) and spleenocytes of mice that were biased by Th2 response. It produced 5.8-8.4 times higher, and suppressed IL-4 production of Th2 reaction-induced cytokin at the level of 10.7-12.9%, which is another typical lactic acid bacterium Lactobacillus rhamnosusGG (KCTC 5033), Lactobacillus casei (KCTC 3109), Lactobacillus sakei CJLS118 (KCTC 13416) was confirmed to be significantly superior. Therefore, Lactobacillus plantarum CJLP56 can be said to have immunomodulatory ability to suppress Th2 response and promote Th1 response, and to regulate Th1 / Th2 imbalance due to excessive Th2 response.

  In addition, Lactobacillus plantarum CJLP56 (Lactobacillus plantarum CJLP56) was treated with macrophage cell line RAW264.7 and dendritic cell JAWSII, resulting in an increase in the number of lactic acid bacteria. It was confirmed that Macrophage cell line RAW264.7 and award cell line JAWSII were treated with Lactobacillus plantarum CJLP56, resulting in Th1 differentiation-inducing cytokin IL-12 and IL-18 production and Th1 differentiation-inducing cytokin IL -10 was confirmed to promote the induction of Th1 differentiation by producing relatively less than the amount of IL-12 produced. Therefore, even from these experimental results, Lactobacillus plantarum CJLP56 has an immunomodulatory ability to suppress Th2 response, promote Th1 response, and regulate Th1 / Th2 imbalance due to excessive Th2 response I can say that.

  IL-4 is an anti-inflammatory cytokine that is derived from Th2 cells and specifically plays a pivotal role in the cellular immune response and suppresses IL-12 production of Th1 cell cytokin Has function. Recently, it is known that Th2 cells mainly producing IL-4, IL-5 and the like are relatively increased in peripheral blood and skin lesions of atopic dermatitis patients (Non-patent Document 10). Thus, Th1 / Th2 imbalance due to excessive Th2 response induces diseases such as atopy. In addition, as previously examined, when one of the Th1 and Th2 reactions is excessive or one of the other is insufficient, the disease is caused, and when the Th1 response decreases and the Th2 response increases, cancer, atopy, It is known that allergies and autoimmune diseases are caused (Non-patent Document 11). Thus, Lactobacillus plantarum CJLP56 in accordance with the present invention regulates Th1 / Th2 imbalance due to Th2 hyperresponsiveness by regulating Thkin, Th2 cells, macropaj and dendritic cells produced by immunomodulation. It is expected not only that it may effectively act against diseases such as atopy and allergy, but is also expected to be effective in the prevention or treatment of cancer and autoimmune diseases.

  Therefore, in another aspect, the present invention provides an immunopotentiating composition comprising Lactobacillus plantarum CJLP56. The composition for enhancing immunity according to the present invention has an immune enhancing effect due to the immune enhancing effect of a general lactic acid bacterium as described in the above-mentioned prior art as Lactobacillus plantarum CJLP56 as a lactic acid bacterium. In particular, in the composition for enhancing immunity according to the present invention, as demonstrated in the Examples below, Lactobacillus plantarum CJLP56 has an effect of promoting Th1 reaction and regulates Th1 / Th2 imbalance due to excessive Th2 reaction Therefore, it is effective for the prevention or treatment of diseases induced by Th1 / Th2 imbalance caused by excessive Th2 response. Therefore, the composition for enhancing immunity according to the present invention can be effectively used for the prevention or treatment of atopy, allergy, cancer and autoimmune diseases. The autoimmune diseases include, but are not limited to, asthma and hay fever.

  The method for administering the composition for enhancing immunity of the present invention is not particularly limited, and it is preferable to administer the composition through the mouth. The dose varies depending on the type of disease that requires immune enhancement, the degree of disease, age, sex, race, therapeutic or preventive purpose, etc. One hundred million animals can be administered.

  The composition for preventing or treating intestinal diseases and the composition for enhancing immunity containing Lactobacillus plantarum CJLP56 according to the present invention contains a lactic acid bacterium that has been proven to be safe, so that there are no side effects and other concerns. It can be used as a cosmetic, feed, or feed additive.

  When the composition according to the present invention is used as a medicine, it can be formulated in a usual pharmaceutical dosage form publicly disclosed in the technical field. The medicament is preferably formulated in a mouthpiece dosage form, for example liquid dosage forms, suspensions, powders, granules, essences, capsules, pills or X-forms. Can be formulated.

  Each of the dosage forms can be prepared by adding a formulation poem and a pharmaceutically acceptable carrier or additive necessary for the production of each dosage form. Typically formulated as a dosage form for oral administration. As a carrier, one or more can be selected and used as diluent, lubricant, binder, disintegrant, sweetener, stabilizer and preservative. One or more kinds of fragrances, vitamins, and antioxidants can be selected and used.

  The carriers and additives can all be pharmaceutically acceptable, specifically lactose, corn starch, soybean oil, rice crystalline cellulose, or mannitol for diluents, magnesium stearate for lubricants. Alternatively, talc and binder are preferably poly (vinylpyrrolidone) or hydroxyl profile cellulose. Also, disintegrants are carboxymethyl cellulose calcium, sodium starch glycolate, porlacryl phosphorus calcium or crospovidone, sweeteners are sucrose, fructose, sorbitol or aspartame, and stabilizers are carboxymethyl cellulose cellulose. Ryumu, β-cyclerodexturin, vitiligo, or Chantan gum, and as preservatives, methyl oxybenzoate, paraoxybenzoic acid profile, or potassium sorbate is preferred.

  In addition to natural ingredients such as plum scent, lemon scent, pineapple scent, hub scent, natural fruit juice, chlorophyllin, flavonoids, natural pigments, fructose, honey, Sugar alcohol, the same sweetening ingredient as sugar, or the same sour agent as oxalic acid and sodium oxalate can be used in combination.

  Non-patent document 11 describes in detail such formulation methods and carriers and additives necessary for formulation.

  The composition according to the present invention can be used as a food. These foods include not only health-functional foods, but also general foods that are widely and regularly consumed every day by humans. When utilized as a health functional food, it can be formulated in the form of a normal health functional food advertised in the art together with a pharmaceutically acceptable carrier or additive. The health functional food can be produced, for example, as an acid agent, granule, essential agent, capsule, suspension, emulsion, syrup, solution, X agent, tea, jelly, or drink. As the food-acceptable carrier or additive, any carrier or additive publicly known to be usable in the art for the production of a dosage form to be manufactured can be used. .

  Since the composition according to the present invention has an effect of preventing or treating atopy, it can also be used as a cosmetic. When the composition according to the present invention is used as a cosmetic, it can be manufactured with various cosmetics in a usual dosage form advertised in the cosmetic technical field. Each of the dosage forms can be prepared by adding a formulation poem and a carrier or additive acceptable for the production of cosmetics necessary for the production of each dosage form.

  The composition according to the present invention can be used as a feed additive or a feed.

  When used as a feed additive, the composition can be produced in the form of a 20 to 90% highly concentrated solution, powder or granules. The feed additive is an organic acid such as oxalic acid, fumaric acid, adipic acid, lactic acid, malic acid, phosphoric acid flame such as sodium phosphate, potassium phosphate, acidic fatigue phosphoric acid flame, polyphosphoric acid flame (polymerized phosphoric acid flame), Contains one or more additional natural antioxidants such as polyphenols, catechins, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, pitinic acid Can do. When used as a feed, the composition is formulated in a normal feed form and can include both normal feed ingredients.

  The feed additive and the feed are cereals such as wheat, oats, wheat, corn and rice pulverized or crushed, for example; vegetable protein feeds such as rape, beans, and sunflower. ; Animal protein feed, blood, meat meal, bone meal and fish for example; sugar and dairy products, dry ingredients made up of various milk powder and milk powder for example, and other nutritional supplements , Digestion and absorption enhancers, growth promoters and the like.

  The feed additive can be administered to animals alone or in combination with other feed additives in an edible carrier. In addition, the feed additive can be easily administered to animals as a top dressing, directly mixed with animal feed, or in a mouthpiece form separate from the feed. When the feed additive is administered separately from animal feed, it can be produced in immediate release or sustained release dosage form in combination with a pharmaceutically acceptable edible carrier as is well known in the art. Such edible carriers can be solid or liquid, such as corn starch, lactose, scroos, bean flakes, peanut oil, olive oil, sesame oil and propylene glycol. If a solid carrier is used, the feed additive can be a essence, capsule, acid, troche or sugar-containing essence or an undispersed form of top dressing. When a liquid carrier is used, the feed additive can be a gelatin soft capsule, or a syrup or suspension, emulsion, or solution dosage form.

  The feed can include any protein-containing organic flour normally used to satisfy an animal's dietary needs. Such protein-containing flours are usually composed primarily of corn, bean flour, or a corn / bean flour mix.

  In addition, the feed additive and the feed may contain auxiliary agents such as preservatives, stabilizers, wetting agents or emulsifiers, solution accelerators, and the like. The feed additive can be used by being added to animal feed after being permeated, sprayed or mixed.

  The feed or feed additive of the present invention can be applied to many animal diets including mammals, poultry and fish. The mammal can be used for pigs, cows, sheep, goats, laboratory animals and laboratory animals, as well as pets (e.g. dogs, cats), etc., and the poultry can be chickens, turkeys, duck, geese, It can also be used for pheasants, quails, and the like, but is not limited to this.

  As described above, the novel lactic acid bacterium Lactobacillus plantarum CJLP56 according to the present invention has excellent acid resistance, bile acid resistance and intestinal epithelial cell adhesion as a probiotic, and has an intestinal regulating effect. It has the effect of promoting the reaction and the effect of regulating the Th1 / Th2 imbalance due to excessive Th2 reaction. Therefore, the novel lactic acid bacterium Lactobacillus plantarum CJLP56 according to the present invention is used as a composition for treating intestinal diseases and a composition for enhancing immunity, and particularly induced by Th1 / Th2 imbalance due to excessive Th2 response. It is effective in preventing or treating diseases.

FIG. 1 is a graph showing the acid resistance test results of Lactobacillus plantarum CJLP56. FIG. 2 is a graph showing the bile acid resistance experimental results of Lactobacillus plantarum CJLP56. FIG. 3 is a graph showing the intestinal epithelial cell adhesion ability test results of Lactobacillus plantarum CJLP56. FIG. 4 shows the results of measurement of IL-12 concentration of Th1 reaction-induced cytokin after treatment of Lactobacillus plantarum strain CJLP56 with spleen splenocytes of mice that were administered ovalbumin and biased by Th2 reaction. It is a graph shown in comparison with lactic acid bacteria. FIG. 5 shows the results of measurement of IL-4 concentration of Th2 reaction-induced cytokin after treatment of Lactobacillus plantarum strain CJLP56 strain with spleen sputum cells that were administered ovalbumin and biased by Th2 reaction. It is a graph shown in comparison with lactic acid bacteria. FIG. 6 is a graph showing the results of measuring the concentration of IL-12 and IL-10 by ELISA after treating the Lactobacillus plantarum CJLP56 strain with the macrophage cell line RAW264.7 and comparing with other lactic acid bacteria. is there. FIG. 7 is a graph showing the results obtained by treating Lactobacillus plantarum CJLP56 strain with dendritic cell line JAWSII and then measuring the concentrations of IL-12 and IL-10 by ELISA in comparison with other lactic acid bacteria. is there. FIG. 8 shows the results of measuring the expression of IL-12p40 and IL-18 mRNA by RT-PCR after treating the Lactobacillus plantarum CJLP56 strain with the macrophage cell line RAW264.7 and comparing it with other lactic acid bacteria. It is a graph to show. FIG. 9 shows the results of measuring the expression of IL-12p40 and IL-18 mRNA by RT-PCR after treating the Lactobacillus plantarum CJLP56 strain with the dendritic cell line JAWSII and comparing it with other lactic acid bacteria. It is a graph to show.

  Hereinafter, the present invention will be described more specifically with reference to the following examples. However, these examples are only for helping understanding of the present invention and do not limit the scope of the present invention in any way.

Experimental Example 1: Isolation and identification of microbial Lactobacillus plantarum CJLP56 strain Lactobacillus plantarum CJLP56 strain isolated by Kimchi was smeared on MRS solid medium (Difco, USA) containing 1.5% agar (agar) The colonies that were confirmed to be purely isolated after culturing at 37 ° C. for 24 hours were taken as platinum (loop) and cultured in MRS liquid medium (Difco, USA) at 37 ° C. for 18-24 hours.

  Next, the morphology and physiological characteristics of the CJLP56 strain were determined using the method of Kim et al. (Non-patent Document 12) and the API50CH and API50CH L kits (Biomerio product). The morphology and physiological characteristics of the CJLP56 strain confirmed as a result are summarized in Table 1 above.

  The base sequence of 16S rRNA gene for lactic acid bacteria identification and classification was analyzed. The method of Kim et al. (Non-patent Document 13) was used to determine and analyze the base sequence of the 16S rRNA gene. The nucleotide sequence of the 16S rRNA gene of CJLP56 confirmed as a result was listed in the sequence listing (SEQ ID NO: 1).

  Lactobacillus plantarum CJLP56 strain showed the highest homology (99.9%) with Lactobacillus plantarum NBRC 15891T, GenBank accession number AB326351 as a result of 16S rRNA nucleotide sequence analysis. It was identified with Lactobacillus plantarm, named Lactobacillus plantarum CJLP56, and deposited with the Biotechnology Institute Genebank on October 16, 2008 (accession number KCTC 11402 BP).

Experimental Example 2: Lactobacillus plantarum CJLP56 strain acid resistance experiment with artificial gastric juice and bile resistance experiment with artificial bile Acid resistance experiment with artificial gastric juice is a modification of the experiment of Kobayashi et al. The artificial gastric juice was prepared using the prepared artificial gastric juice. Specifically, the artificial gastric juice was prepared by sterilizing the MRS liquid medium with 1N HCl to pH 2.5 and adding pebcine to 1000 units / ml. .

Lactobacillus plantarum CJLP56 isolated and identified from Example 1 above was cultured in an MRS liquid medium at 37 ° C. for 18 hours to centrifuge the lactic acid bacteria to precipitate, and sterile saline (0.85 After rinsing twice with (NaCl), the bacterial cell suspension was inoculated into the control medium and artificial gastric juice at a level of about 10 ⁷ cfu / ml, and cultured at 37 ° C. Was measured. The total number of bacteria was measured by diluting 10 times with a phosphate buffer (pH 6.8) containing KH ₂ PO 4, Na ₂ HPO, L-cysteine, HCl, Tween 80 and the like.

  The bile resistance experiment with artificial bile progressed by the method of Casey et al. (Non-patent Document 15). After 0.3% of bull bile was added to the MRS liquid medium used in the acid resistance evaluation, the acid resistance evaluation method The number of viable bacteria was measured after 0, 12, and 24 hours after inoculation with lactic acid bacteria.

  The same comparative experiment was conducted for the above-mentioned acid resistance evaluation and bile acid resistance evaluation for Lactobacillus casei (KCTC 3109), Lactobacillus sakei CJLS118 (KCTC 13416), and Lactoba ilus rhamnosus GG (KCTC 5033). Carried out.

  The results are shown in FIG. 1 and FIG. FIG. 1 is a graph showing the results of acid resistance experiments of Lactobacillus plantarum CJLP56. FIG. 2 is a graph showing the results of bile acid resistance experiment of Lactobacillus plantarum CJLP56.

  From the results of FIGS. 1 and 2, it was revealed that Lactobacillus plantarum CJLP56 has acid resistance and bile acid resistance equal to or higher than those of all other lactic acid bacteria subjected to the comparative experiments. This indicates that the novel strain according to the present invention can reach the intestine without being affected by gastric juice in the body and can survive without being affected by bile in the intestine.

Example 3: Lactobacillus plantarum CJLP56 strain adhesion test for intestinal epithelial cells HT-29 was used as an animal cell for intestinal epithelial cell adhesion test and was used at the Korea Cell Line Bank (KCLB), The experimental method used was Kim et al. (Non-Patent Document 16) and Hirono et al. (Non-Patent Document 17).

HT-29 cells are RPMI 1640 (Gibco) supplemented with heat-inactivated 10% fetal bovine serum (FBS), 1% L-glutamine, penicillin G (100 IU / mL), and streptomycin (100 mg / mL). , USA) medium and cultured at 37 ° C. in the presence of 5% CO ₂ . For adhesion and adhesion inhibition experiments, HT-29 cells were divided into 24-well plates as much as 1.0 × 10 ⁵ cells / mL per well, and the medium was changed every other day. It was cultured until the time when a monolayer was formed and used for the experiment. HT-29 cells that formed a complete monolayer were washed 5 times using a PBS buffer solution at 25 ° C., and 0.5 mL of RPMI 1640 medium to which no antibiotic was added was added.

  Lactobacillus plantarum CJLP56 was suspended in RPMI to a concentration of about 1.0 × 109 cfu / mL, inoculated into the well plate, and cultured at 37 ° C. for 2 hours in the presence of 5% CO2. . Washing was carried out 3 times using PBS buffer solution with stirring at a rate of 200 rpm for 3 minutes in order to confirm the adherence ability accompanying removal and washing of lactic acid bacteria that did not adhere after completion of the culture. After washing is completed, 0.2% trypsin-EDTA is separated to separate the attached cells, and the MRS-agar is smeared in a serial dilution method using the peptone number. The cells were cultured for 24 hours and the number of bacteria was measured.

  Separately, in order to confirm partial adhesion, a cover glass soaked in 70% alcohol for about a day and completely sterilized is attached to the bottom of the petrity tissue, and then the HT-29 cells are cultured, and the same amount of lactic acid bacteria is added. The experiment was conducted with addition. Lactic acid strains that were not washed away by washing and adhered to HT-29 cells were dried and then stained with Gram and observed with an optical microscope to determine the number of bacteria. The same comparative experiment was performed for Lactobacillus sakei CJLS118 and Lactobaillus rhamnosusGG (KCTC 5033).

  The results are shown in FIG. FIG. 3 is a graph showing the intestinal epithelial cell adhesion ability test results of Lactobacillus plantarum CJLP56.

  According to the results in FIG. 3, Lactobacillus plantarum CJLP56 adheres to intestinal epithelial cells after 24 hours compared to Lactobacillus rhamnosusGG (KCTC 5033) and Lactobacillus sakei CJLS118, which are well known commercially in probaotic strains. It was revealed that the ability of the intestinal epithelial cells was significantly higher than that of Lactobacillus sakei CJLS118. These results indicate that the novel strain according to the present invention may adhere to intestinal epithelial cells and improve the intestinal environment.

Example 4: Safety evaluation of Lactobacillus plantarum CJLP56 strain In order to evaluate the safety of the strain isolated in Example 1, hemolysis phenomenon test by the safety evaluation test method presented in the Korea Bioventure Association group standard Gelatin liquefaction test, harmful metabolite (ammonia) production confirmation, peninalanine taramine test were performed.

  The results are shown in Table 2 below.

  According to the above results, Lactobacillus plantarum CJLP56 (Lactobacillus plantarum CJLP56) is negative for gelatin liquefaction, harmful metabolite (ammonia) production, peninalanine taramine production, pathogenicity in hemolysis phenomenon test It was confirmed by α-hemolysis determined to be unrelated. Therefore, Lactobacillus plantarum CJLP56 was confirmed to be a safe strain that can be administered to the human body.

Example 5: Evaluation of IL-12 production promoting ability after treatment with mouse spleen sputum cells In order to evaluate the IL-12 production promoting ability of reaction-induced cytokin, an experiment was conducted as follows with reference to Fujiwara et al. (Non-patent Document 18) and Fujiwara et al.

For immunization, 5 6-week-old female Balb / c mice were purchased and mixed well with alum hydroxide (Sigma) 13 mg / mL solution 1.538 mL, ovalbumin 10 mg and PBS 0.4615 mL at room temperature. The mixed liquid reacted for 1 minute was injected into the peritoneal cavity by 0.2 mL (1 mg OVA + 2 mg alum) per mouse, and the same amount was boosted by injection into the abdominal cavity on the sixth day. The mouse was sacrificed on the 13th day and the splenic sputum was removed and the splenocyte obtained from this was 100 μl (4 × 10 ⁶ cells / mL), 50 μl of the killed bacteria of the test organism and 50 μl of egg albumin (4 mg / ml) Was added to a cell culture well plate and cultured in a DMEM-10 medium for 7 days in a 10% CO ₂ incubator. After culturing for 7 days, the supernatant was removed and assayed (Biosource) with the IL-12 ELISA kit to measure the concentration of IL-12.

  The dead bacteria of the test target bacteria were obtained as follows.

  The test bacteria were inoculated into MRS liquid medium (Difco), cultured at 37 ° C for 24 hours, and centrifuged at 13,000 rpm for 1 minute. . The collected cells were heated in sterile distilled water of the same volume as the original culture volume for 10 minutes at 100 ° C for 10 minutes and centrifuged at 13,000 rpm for 1 minute, and collected to DMEM medium. An appropriate amount was diluted so as to obtain cells having a concentration of 50 μg / ml and 5 μg / ml as a cell line culture volume reference. Lactobaillus rhamnosus GG (KCTC 5033), Lactobacillus casei (KCTC 3109), and Lactobacillus sakei CJLS118 (KCTC 13416) were also used as the test bacteria. Compared.

  The IL-12 assay (assay) using the IL-12 ELISA kit was performed according to the instructions provided in the IL-12 ELISA kit, and the OD value measured by the ELISA reader was measured and provided to the kit. The IL-12 production amount was converted by a calibration formula for the IL-12 control sample. The measurement results thus obtained are shown in FIG.

  FIG. 4 shows the results of measurement of IL-12 concentration of Th1 reaction-induced cytokin after treatment of Lactobacillus plantarum strain CJLP56 with spleen splenocytes of mice that were administered ovalbumin and biased by Th2 reaction. It is the graph shown in comparison with lactic acid bacteria.

  The results of FIG. 4 revealed that Lactobacillus plantarum CJLP56 significantly promoted the production of IL-12, a Th1 reaction-induced cytokin, compared to other lactic acid bacteria. Therefore, it was confirmed that Lactobacillus plantarum CJLP56 according to the present invention effectively induces Th1 response in mice biased by Th2 response.

Example 6: Evaluation of inhibitory ability of IL-4 production after treatment of mice splenomegaly cells In order to confirm the inhibitory effect of reaction-induced cytokin on IL-4 production, the remaining conditions were the same except that the IL-4 kit (Biosource) was used instead of the IL-12 kit in the method of Example 5. The experiment proceeded. The measurement results are shown in FIG.

  FIG. 5 shows the results of measurement of IL-4 concentration of Th2 reaction-induced cytokin after treatment of Lactobacillus plantarum strain CJLP56 strain with spleen sputum cells that were administered ovalbumin and biased by Th2 reaction. It is the graph shown in comparison with lactic acid bacteria.

  According to the results shown in FIG. 5, Lactobacillus plantarum CJLP56 has an inhibitory effect on Th2 response in mouse splenic sputum cells biased by Th2 response by suppressing IL-4 cytokin of Th2 response-induced cytokin. I was able to confirm that there was.

Example 7: Expression of Th1 lymphocyte differentiation-inducing cytokin IL-12p40 and IL-18 in macrophages and dendritic cells, and IL-10 expression confirmation experiment of Th1 lymphocyte differentiation-inhibiting cytokin Antigens such as macrophages and dendritic cells The presenting cell (antigen presenting cell, APC) produces IL-12 and IL-18 and induces differentiation of Th0 lymphocytes with Th1 lymphocytes, while the other produces IL-10 and Th1 lymphocytes. Suppresses differentiation induction. In order to evaluate the effect of lactic acid bacteria according to the present invention on the production of IL-12, IL-10, and IL-18 by macrophages and dendritic cells, the following experiment was performed.

The test bacteria were treated with RAW264.7, a macrophage cell line, at a concentration of 5 × 10 ⁷ / mL and 37 ° C., 10% CO ₂ culture was performed for 48 hours. The concentration of IL-10 was measured. In addition, the dendritic cell line JAWSII was inoculated and cultured in the same manner, and then the culture medium was taken and the production amounts of IL-12p40 and IL-10 were measured.

  Lactobacillus plantarum CJLP56 was used as the test microorganism, and lipopollisacaride was used as a control group, and Lactobaillus rhamnosus GG (KCTC 5033), Lactobacillus casei (KCTC 3109), Lactobacillus sakei CJLS118 The same experiment was performed on (KCTC 13416) and the results were compared.

  The concentration measurement by the ELISA method is produced using an IL-12p40 kit (BD Biosciences, USA) capable of measuring the concentration of IL-12 and an IL-10 kit (BD Biosciences, USA) capable of measuring the concentration of IL-10. Performed according to company guidelines. The results thus measured are shown in FIG. 6 and FIG.

  FIG. 6 is a graph showing the results of measuring the concentration of IL-12 and IL-10 by ELISA after treating the Lactobacillus plantarum CJLP56 strain with the macrophage cell line RAW264.7 and comparing it with other lactic acid bacteria. It is.

  FIG. 7 is a graph showing the results of measuring the concentration of IL-12 and IL-10 by ELISA after treating the Lactobacillus plantarum CJLP56 strain with the dendritic cell line JAWSII and comparing it with other lactic acid bacteria. It is.

  According to the results of FIGS. 6 and 7, Lactobacillus plantarum CJLP56 generates Th-12 differentiation-inducing cytokin IL-12 cytokin, and Th1 differentiation-inhibiting cytokin IL-10 is produced by IL-12. It can be confirmed that the production is significantly less than that of lactic acid bacteria, and that the production of IL-12 is significantly increased compared to other lactic acid bacteria.

  In addition, in order to confirm the production of IL-12 and IL-18 at the gene level, the test bacteria were treated with RAW264.7 of the macrophage cell line at a concentration of 5 × 10 7 / mL at 37 ° C., 10 After performing% CO2 culture for 6 hours, total RNA was extracted, and the production amounts of IL-12 and IL-18 mRNA were measured by RT-PCR method. The dendritic cell line JAWSII was inoculated and cultured by the same method, and RNA was extracted, and the production of IL-12 and IL-18 mRNA was measured by RT-PCR.

  The results thus measured are shown in FIG. 8 and FIG.

  FIG. 8 shows the results of measuring the expression of IL-12p40 and IL-18 mRNA by RT-PCR after treating the Lactobacillus plantarum CJLP56 strain with the macrophage cell line RAW264.7 and comparing it with other lactic acid bacteria. It is the shown graph.

  FIG. 9 shows the results of measuring the expression of IL-12p40 and IL-18 mRNA by RT-PCR after treating the Lactobacillus plantarum CJLP56 strain with the dendritic cell line JAWSII in comparison with other lactic acid bacteria. It is a graph.

  8 and 9, it can be seen that Lactobacillus plantarum CJLP56 promotes the production of mRNA that directs the production of IL-12 and IL-18 of the Th1 differentiation-inducing cytokin, It was confirmed that IL-12 mRNA expression was significantly better than other lactic acid bacteria.

Example 8: Production of probiotic agent containing Lactobacillus plantarum CJLP56 (Lactobacillus plantarum CJLP56) Probiotic Lactobacillus plantarum CJLP56 (Lactobacillus plantarum CJLP56) identified in Example 1 They were mass-produced for application with feeds, feed additives, or cosmetic ingredients, and lyophilized into viable bacteria.

  For the production of the bacteria, the cells were collected by culturing at 37 ° C. for about 18 hours while adjusting the pH to 6.0 with MRS liquid medium (Difco) using a 25% NaOH solution and collecting the cells. The collected cells were frozen using lyoprotectant with 5% dextrin and 10% nonfat milk and frozen at -40 ° C, then dried at 37 ° C and crushed with a mixer. The separated viable bacteria were packaged in a sealed aluminum pouch mixed with the same excipient as sucrose, lactose, skim milk, etc. in order to store them according to the target number of bacteria.

  The live bacteria produced in this way is mixed with grain flour used as a raw material in feed and used in live feed for feed, or mixed with carriers or additives, etc. It can be used in a variety of fields such as pharmaceuticals, foods, feeds, cosmetics and the like by ordinary methods in the technical field by using them in live fungi or by mixing a certain amount with raw materials used in cosmetics.

Claims (9)

  Lactobacillus plantarum CJLP56 (KCTC 11402BP).   Lactobacillus plantarum CJLP56 (Lactobacillus plantarum CJLP56) The composition for the prevention or treatment of an intestinal disease containing KCTC 11402BP.   Lactobacillus plantarum CJLP56 (Lactobacillus plantarum CJLP56) The composition for immune enhancement containing KCTC 11402BP.   The composition according to claim 3, which is used for prevention or treatment of an immune disease induced by Th1 / Th2 imbalance due to an excessive Th2 reaction.   The composition according to claim 4, wherein the immune disease is selected from the group consisting of allergic diseases, atopy, cancer and autoimmune diseases.   The composition according to claim 2, wherein the composition is a pharmaceutical product.   The composition according to any one of claims 2 to 5, wherein the composition is a food.   The composition according to any one of claims 2 to 5, which is a feed or a feed additive.   The composition according to claim 2, wherein the composition is a cosmetic.

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